| The occurrence of gastric cancer is a complex biological process.At present,the common recognized mode of the occurrence of GC is "chronic non-atrophic gastritis chronic atrophic gastritis→intestinal metaplasia→ dysplasia→GC" proposed by Correa.The intestinal metaplasia and dysplasia associated with chronic atrophic gastritis is called gastric precancerous lesions(GPL).Researches have shown that early intervention at GPL stage can effectively block the occurrence of GC.At present,modern medicine still lacks specific treatment for GPL,while studies have shown that traditional Chinese medicine has its unique advantages in blocking and reversing GPL.In addition,it has the characteristics of multi-target and safe use,thus attracted widespread attention.Meanwhile,scholars have also conducted a lot of in-depth exploration on the mechanism of traditional Chinese medicine in the treatment of GPL.Uncontrolled inflammation is one of the main factors in the occurrence and development of GPL,which can promote the malignant progression of gastric mucosa through various mechanisms.How to reasonably regulate the inflammatory response of gastric mucosa is the top priority in blocking the "inflammation-cancer transformation" of the stomach.Macrophages pyroptosis,as an inflammatory mode of cell death,plays an indispensable role in the formation of local inflammatory immune microenvironment.It is closely related to the occurrence and development of various inflammatory diseases,but its role in GPL has not been elucidated.After long-term clinical observation,our research group proposed that“spleen deficiency,blood stasis and toxin entrapment”is the core pathogenesis of GPL,and the Jianpi Huayu Jiedu Method is the basic principle for treating GPL,based on which,we developed Jianpi Huayu Jiedu Formula(JPHYJD).Clinical researches have shown that it can improve the symptoms of GPL patients and gastric mucosal lesions effectively.Our research group have proved that the molecular mechanism of JPHYJD in treating GPL closely related to inhibition of inflammation.Therefore,our study intends to explore the role of macrophage pyroptosis in GPL and clarify the mechanism of JPHYJD in treating GPL from the perspective of macrophage pyroptosis.Objective1.GPL models were constructed in vitro and in vivo,and intervened by JPHYJD to observe the efficacy of JPHYJD in the treatment of GPL2.Infiltration of macrophages in gastric mucosa of GPL patients and mice was observed to explore the correlation between macrophages and GPL lesions and the inhibitory effect of JPHYJD;3.Pyroptosis of macrophage in gastric mucosa of GPL mice were observed and the macrophage pyroptosis cell models were constructed to explore the correlation between macrophage pyroptosis and GPL and the inhibitory effect of JPHYJD;4.To explore the molecular mechanism of JPHYJD in treating GPL by regulating PKA/NLRP3 signal pathway and inhibiting pyroptosis.Methods1.Construction of GPL mice and cell models and the evaluation of the effect of JPHYJDC57BL/6 mice were provided with free drinking MNU solution and were subjected to feed every other day to construct GPL model.Then the mice were intervened by different dosed of JPHYJD and vitacoenzyme respectively.HE staining and AB-PAS staining were used to observe the histopathological changes of gastric mucosa;electron microscopy was used to observe the ultrastructural changes;Immunohistochemistry was sued to detect the expression of CDX2 and P53.Prepare the JPHYJD containing serum.GES-1 cells were exposed to 100μg/mL MNU for 48h to construct GPL cell models and intervened by JPHYJD containing serum.EdU was used to examine cell proliferation and western blot to examine the expression of Bcl-2,Bax,CD44,PTEN,Survivin,STAT3、P-STAT3、NF-κB、P-NF-κB;2.The infiltration of macrophage in GPL mucosa of patients and mice modelsImmunohistochemistry(IHC)was used to detect the expression of macrophage markers CD68 and CD 14 in normal gastric mucosa,chronic atrophic gastritis,intestinal metaplasia,dysplasia and GC;Number of macrophage in gastric mucosa of GPL mice was detected by flow cytometry;Immunofluorescence staining(IF)was used to observe the expression of CDX2,Ki67 and macrophage marker F4/80 in gastric mucosa of GPL mice;3.Construction of macrophage pyroptosis models and the interventional effect of JPHYJDIHC,WB,PCR and IF were used to detect the expression of pyroptosis-related molecules such as NLRP3,GSDMD,GSDMD-N,ASC,Caspase-1,IL-1β;IF was used to observe the colocation of F4/80 and NLRP3 in gastric mucosa of GPL mice;J774A.1 and THP-1 were exposed to LPS and ATP to construct pyroptosis models,and the JPHYJD containing serum was used for intervention.WB,PCR,and IF were used to examine the expression of pyroptosis related molecules;Caspase-1 activity test kit was used to detect Caspase-1 activity of cells in each group;PI-Hoechst double staining was used to observe the pyroptosis of macrophages.4.PKA/NLRP3 mediates the occurrence of macrophage pyroptosis and the interventional effect of JPHYJDMacrophages were divided into control group,model group,JPHYJD group,H89 group,model+H89 group and model+H89+JPHYJD group.On the basis of construction of pyroptosis cell models,H89 were used as PKA inhibitor.WB was used to determine the expression of PKA,NLRP3,GSDMD and IL-1β;The level of pyroptosis was observed by PI-Hoechst staining.Mice were divided into control group,model group,JPHYJD group,H89 group,model+H89 group and model+H89+JPHYJD group.Based on the construction of GPL mouse models,H89 was used as PKA inhibitor in corresponding group intraperitoneally.WB was used to determine the expression levels of GSDMD-N,Caspase-1 p10 and IL-1β.HE staining and AB-PAS staining were used to observe the pathological changes of gastric mucosa of mice in each group.Results1.Construction of GPL mice and cell models and the evaluation of the effect of JPHYJDIn vivo,HE staining showed that compared with the control group,GPL signs were observed in mucosa of mice in model group such as thinner gastric mucosa,flattened mucosa folds,decreased number of glands and irregular arrangement,and uneven size of epithelial cells and lymphocyte infiltration,etc;AB-PAS staining showed that in model group,the part near lamina propria of gastric mucosa was stained blue and purple,indicating small intestinal metaplasia.Transmission electron microscopy showed that cells of gastric mucosa in model group were discontinuous with surrounding structures,and the polarity disappeared,the glandular cavity turned into pseudostratified epithelium;the adjacent basement membrane fused and dissolved,and the adjacent epithelial cells connected significantly(back-to-back phenomenon),and the single epithelium turned into pseudostratified epithelia;abnormal vascular endothelial cells also could be observed.IHC showed that the expression level of CDX2 and P53 increased in the model group(P<0.01).Both high dose and low dose of JPHYJD and vitacoenzyme could improve the pathological changes and ultrastructure of mucosa.and decrease the expression level of CDX2 and P53(P<0.01).In vitro,GES-1 cells showed irregular shape,high proliferation activity and expression of GPL markers,inhibition of apoptosis,activation of STAT3 and NF-κB after MNU intervention,indicating that GPL models were constructed successfully.EdU staining showed that MNU significantly increased the proliferative activity(P<0.01),which could be decreased by JPHYJD(P<0.01).The level of Bax/Bcl-2 decreased,while the level of CD44,PTEN,Survivin,P-NF-κB/NF-κB and P-STAT3/STAT3 increased in model group as was shown by WB(P<0.01),and also be regulated by JPHYJD(P<0.01).2.The infiltration of macrophage in GPL mucosa of patients and mice modelsThe results of IHC showed that the average optical density of CD68 and CD 14 in gastric mucosa of patients with intestinal metaplasia dysplasia and GC increased compared with the normal group(P<0.01);Flow cytometry showed that compared with the control group,the proportion of macrophage in gastric mucosal in model group increased(P<0.05);IF showed that the expression level of CDX2,Ki67 and F4/80 increased in model group(P<0.01),and were reduced by high dose and low dose of JPHYJD and vitacoenzyme(P<0.01).3.Construction of macrophage pyroptosis models and the interventional effect of JPHYJDThe results of IHC,WB,PCR and IF staining showed that pyroptosis related molecules like NLRP3,Caspase-1,GSDMD-N,IL-1β and IL-18 in model group increased(P<0.01,P<0.05),and different dose of JPHYJD and vitacoenzyme could reduce the expression level(P<0.01,P<0.05);IF staining showed the colocalization of F4/80 and NLRP3 upregulated.JPHYJD could downregulate the colocalization of F4/80 and NLRP3 in GPL mucosa.In vitro,pyroptosis related molecules such as NLRP3,Caspase-1,GSDMD-N,IL-1β,IL-18 were upregulated in model group(P<0.01,P<0.05)and also the Caspase-1 activity(P<0.01)and the proportion of PI staining positive cells(P<0.01),which were all downregulated by JPHYJD(P<0.01,P<0.05).4.PKA/NLRP3 mediates the occurrence of macrophage pyroptosis and the interventional effect of JPHYJDIn vitro,the level of PKA decreased and the level of NLRP3,GSDMD and IL-1βincreased in model group and model+H89 group than in the control group(P<0.01),which was more significant in model+H89 group(P<0.01);while JPHYJD could upregulate PKA and downregulate NLRP3,GSDMD and IL-1β(P<0.01),while the effect could be partly blocked by H89(P<0.01).PI staining showed that in model group and model+H89 group,the proportion of PI staining positive cells increased(P<0.01)and more significantly in model+H89 group(P<0.01).JPHYJD could decrease the proportion(P<0.01),while in model+H89+JPHYJD group there was no difference compared with the model group(P>0.05);In vivo,compared with the control group,the expression level of Caspase-1 p10,GSDMD-N and IL-1β increased in model group and model+H89 group(P<0.01),and the level in model+H89 group was higher than that in model group(P<0.01,P<0.05);compared with the model group,JPHYJD could downregulate the level of Caspase-1 p10,GSDMD-N and IL-1β(P<0.01);however the effect could be blocked by H89 to a certain degree.HE staining showed that compared with the control group,GPL signs of thinner mucosa,thicken basement membrane,less glands with structural disorder and irregular arrangement were observed in model group and model+H89group,and the results of AB-PAS staining indicated intestinal disorder.The lesion degree of model+H89 group was more severe than that of model group.JPHYJD could improve the lesion of GPL,but not in the model+H89+JPHYJD group.Conclusion1.JPHYJD could significantly improve the gastric mucosal lesions of GPL mice,inhibit the expression of molecular markers of GPL,and inhibit the malignant transformation of GES-1 cells induced by MNU;2.Macrophage infiltration is involved in the progression of GPL,and JPHYJD could reduce the infiltration of macrophage in GPL gastric mucosa;3.The level of macrophage pyroptosis increased in gastric mucosa in GPL mice,and the effect of JPHYJD may be related to its inhibition of macrophage pyroptosis;4.JPHYJD could inhibit macrophage pyroptosis through the regulation on PKA/NLRP3 signal pathway,thereby play a role in treating GPL. |
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